Protein arginine methyltransferases (PRMTs) are eukaryotic enzymes that transfer a methyl group from S-adenosylmethionine (SAM) to the guanidino nitrogen of an arginine residue to form monomethyl arginine (MMA) as shown in FIG. 1. All PRMTs generate MMA, however they are classified as either type 1 or type 2, depending on the type of dimethylated arginine that they form. Type 1 PRMTs produce asymmetric dimethyl arginine (ADMA) upon the second round of methylation, whereas type 2 isozymes generate symmetric dimethyl arginine (SDMA). To date, eleven putative and confirmed PRMTs have been described in humans. PRMTs 1, 3, 4, 6, and 8 exhibit type 1 activity, while PRMTs 5, and 7 have type 2 activity. PRMTs 2 and 10 have not yet been shown to possess either type 1 or type 2 activity, thus they remain unclassified. Interestingly, PRMTs 9 and 11 possess catalytic domains that lack significant homology to the catalytic core domains of the other PRMT family members; thus these isozymes may not represent bona fide PRMTs.
PRMT1 is the predominant PRMT in mammalian cells and is thus responsible for the majority of the arginine methyltransferase activity in vivo. This enzyme is well conserved, both structurally and functionally, among eukaryotes. The human orthologue is composed of 353 amino acids (40.5 kDa) that primarily make-up the catalytic core. The structure of PRMT1 has been determined by X-ray crystallography and contains a Rossmann-type fold in the N-terminal half of the protein; this fold is involved in SAM binding as illustrated in FIG. 2. The C-terminal portion of the enzyme contains a consensus region, unique to PRMTs, and forms a β-barrel type fold that is thought to aid substrate binding. There is also a 3-helix ‘arm’ domain that protrudes from the C-terminal region of the protein; this arm is able to interact with the SAM binding domain of a second PRMT monomer to form a head-to-tail homodimer. Studies have demonstrated that the enzyme is only catalytically active in its dimeric form. Deletion or mutation of the helical arm prevents dimerization and results in catalytically inactive monomeric enzymes. Although the crystal structure of the enzyme is available, there is not a high-resolution structure of an enzyme:substrate complex in which residues N- and C-terminal to the site of methylation can be discerned. For this reason, the particular residues involved in substrate binding have yet to be identified.
PRMT1 was originally identified through its interactions with TIS21 and BTG1 proteins and the interferon-alpha receptor. The enzyme has since been implicated in an array of biological processes, including RNA metabolism, protein trafficking, cellular differentiation, and nuclear receptor mediated gene transcription. Current studies have primarily focused on the coactivator activity of the enzyme. In particular, PRMT1 has been found to interact with a number of transcription factors and transcriptional coactivators, e.g. p300/CBP, p53, YY1, and NF-κB, and coactivate transcription by methylating arginine 3 of histone H4.
Although PRMT1 is involved in cellular signaling, its aberrant activity has been implicated in heart disease and cancer. As the major PRMT in vivo, PRMT1 produces the majority of cellular ADMA. Upon proteolysis, free ADMA is released and competes with L-arginine for binding to nitric oxide synthases, thereby inhibiting these enzymes. This results in a decrease in the amount of NO, an important cell signaling molecule that increases vasodilation as illustrated in FIG. 3. Patients suffering from atherosclerosis, hypercholesterolemia and congestive heart failure have elevated levels of ADMA in the plasma and also show an increased expression of PRMT1. This seemingly causal relationship is further bolstered by studies with mice that are incapable of synthesizing dimethylarginine diaminohydrolase 1 (DDAH1). These knock-out mice exhibit increased serum levels of ADMA, reduced NO signaling, elevated systemic and pulmonary blood pressure and endothelial dysfunction. Apparently, if the elevated levels of ADMA could be decreased, then the synthesis of NO would be increased, leading to improved vascular homeostasis.
Likewise, excessive PRMT1 activity has recently been implicated in breast cancer. Hypermethylated ERα has been observed in some breast tumors, suggesting that the dysregulation of ERα methylation, may be involved in breast cancer development [15]. PRMT1 is known to interact with and regulate the transcriptional activity of the estrogen receptor [16]. Le Romancer et al. have demonstrated that hormone binding to the estrogen receptor stimulates PRMT1 to methylate ERα, primarily at arginine 260 in vitro and in vivo [15]. Concurrently, FAK (focal adhesion kinase-1) is partially dephosphorylated, inducing its interaction with the tyrosine kinase, Src, and subsequently the formation of a macromolecular complex, composed of ERα, Src, FAK and p85. This complex formation activates the protein kinase Akt (also known as protein kinase B), which is involved in cellular survival pathways. The indirect upregulation of Akt by PRMT1 activity is thought to be involved in the survival and proliferation of breast cancer cells. Thus, inhibitors that potently and selectively inhibit PRMT1 could potentially be used to regulate PRMT1 methylation activity, and serve as therapeutics for heart disease and breast cancer.
As such, a need exists for an irreversible inhibitor, targeting protein arginine methyltransferase 1.